Microbiology testing laboratories maintain in-house culture collections of microorganisms for quality control purposes. These microorganisms are known as quality control (QC) strains and are used as reference standards and for quality control of the testing methods. Samples that are known to be free of contamination are often spiked with a QC strain and this spiked sample is then processed through the test method. A positive result from the spiked sample validates the testing method. The QC strains are also used to quality control media that is used to grow microorganisms. The media is inoculated with the QC strain and the growth is observed.
Traditionally these quality control checks that are performed with QC strains are qualitative. Recently, however, regulatory authorities such as ISO have begun enforcing quantitative quality control checks.
One problem that microbiology laboratories face is the issue of cross contamination. Laboratories can inadvertently contaminate a real sample with a QC strain. This results in a false positive result, which can have enormous implications such as unnecessary product recalls or incorrect diagnosis of disease.
In order to help with identifying instances of cross contamination, laboratories try to use species of bacteria as QC strains that are rarely detected in their samples. For example, in Australia Salmonella salford is used as a QC strain because it is rarely detected in clinical, food or environmental samples. When a laboratory detects Salmonella, tests are performed to check that the Salmonella detected is not Salmonella salford. If it does prove to be Salmonella salford then the validity of the result is questioned.
The use of rare species such as Salmonella salford as QC stains does help to identify cross contamination problems, however, confirming the identity of the strain that has been detected takes time. Typically, this confirmation takes between one and three days. In some instances, the confirmation has to be performed by a specialist laboratory. These lengthy delays can have serious implications. For example, a product recall may be delayed for several days during which time consumers would be exposed to the risk of infection.
A further problem with the use of rare species as QC strains is that the rare species may have biochemical or physiological properties that are different to those of the commonly isolated organisms. For example, Salmonella salford does not grow well on some media that are routinely used to isolate Salmonella from food, whereas the commonly isolated Salmonella such as Salmonella typhimurium do grow well on these media. Salmonella salford is therefore not a suitable QC strain for these culture media.
In an attempt to address this problem, the present inventors hypothesized that it would be extremely useful to have QC microorganisms that form colonies on agar plates are fluorescent when viewed by the naked eye with illumination from a UV lamp.
The genetic modification of microorganisms with fluorescent genes has been widely studied (GFP: Properties, Applications, and Protocols (1998) Chalfie M, Kain S. Wiley-Liss, New York, USA). The most commonly employed gene for a fluorescent protein is the green fluorescent protein (GFP) gene (gfp) from the jellyfish Aequorea victoria. Genes encoding other fluorescent proteins have also been isolated from other coelenterates.
Fluorescent bacteria have been created previously by incorporating a gfp gene into a plasmid and inserting the plasmid into the bacteria. The plasmid normally contains an antibiotic resistant gene that allows the bacteria to be grown on antibiotic containing media. The antibiotics kill any bacteria that do not retain the plasmid. These plasmid-containing strains only retain their fluorescence when grown on media that contain antibiotics.
An advantage of plasmid-carrying strains is that several hundred copies of the plasmid are normally present within a bacterial single cell. This means that several hundred copies of the fluorescence gene can be placed within each cell to create cells that are very fluorescent.
Plasmid instability can be a major problem in culturing bacteria, particularly if the cultures go through many generations by passaging. The resulting effects are loss of expression of any plasmid-encoded phenotype because of the build-up of non-productive plasmid-free cells. Plasmid instability can be due to segregational instability and/or structural instability. Segregational instability is the loss of plasmid from one of the daughter cells during cell division because of defective partitioning. Structural instability is attributed to deletions, insertions and rearrangements in the plasmid DNA, resulting in the loss of expression of the encoded phenotype. Plasmid stability is influenced by the vector and host genotypes, vector copy number, and the origin and size of foreign DNA have been observed to affect plasmid stability. Plasmid stability is also a function of physiological parameters that affect the growth rate of the host cell, which include pH, temperature, aeration rate, medium components and heterologous protein accumulation.
Plasmid instability is undesirable in the production of bacterial strains for quantitative QC methods, as consistent expression of the QC phenotype is paramount. Consistent expression could be achieved by irreversibly integrating the genes encoding the fluorescent phenotype into the host genome to ensure long-term stability and expression of the gene product. Ideally, only a single copy of the marker gene should be integrated into the bacterial chromosome as this reduces the likelihood of gene instability resulting from homologous recombination-mediated gene excision.
The preferred requirement of a single copy fluorescence gene in the bacterial genome means that achieving sufficient fluorescence maybe challenging. In comparison, the use of a plasmid containing strain allows several hundred copies of the fluorescence gene to be present. To ensure that a high level of fluorescence is achieved with a single copy on the genome a transcriptional promoter should be chosen that is powerful enough to produce visible fluorescence.
It has been found to be difficult to incorporate genes into a bacterial chromosome and still obtain the required selective or characteristic genotype.
The present inventors have developed several strong bacterial promoter systems for the expression of fluorescent phenotypic markers in microbial cells.